Local persistence of novel regional variants of La Crosse virus in the Northeast United States Gillian Eastwood ( [email protected] ) University of Leeds https://orcid.org/0000-0001-5574-7900 John J Shepard Connecticut Agricultural Experiment Station Michael J Misencik Connecticut Agricultural Experiment Station Theodore G Andreadis Connecticut Agricultural Experiment Station Philip M Armstrong Connecticut Agricultural Experiment Station Research Keywords: Arbovirus, Vector, Mosquito species, La Crosse virus, Pathogen persistence, Genetic distinction, Public Health risk Posted Date: October 14th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-61059/v2 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on November 11th, 2020. See the published version at https://doi.org/10.1186/s13071-020-04440-4. Page 1/16 Abstract Background: La Crosse virus [LACV] (genus Orthobunyavirus, family Peribunyaviridae) is a mosquito- borne virus that causes pediatric encephalitis and accounts for 50-150 human cases annually in the USA. Human cases occur primarily in the Midwest and Appalachian regions whereas documented human cases occur very rarely in the northeastern USA. Methods: Following detection of a LACV isolate from a eld-collected mosquito in Connecticut during 2005, we evaluated the prevalence of LACV infection in local mosquito populations and genetically characterized virus isolates to determine whether the virus is maintained focally in this region. Results: During 2018, we detected LACV in multiple species of mosquitoes, including those not previously associated with the virus. We also evaluated the phylogenetic relationship of LACV strains isolated from 2005-2018 in Connecticut and found that they formed a genetically homogeneous clade that was most similar to strains from New York State. Conclusion: Our analysis argues for local isolation and long-term persistence of a genetically distinct lineage of LACV within this region. We highlight the need to determine more about the phenotypic behavior of these isolates, and whether this virus lineage poses a threat to public health. Background La Crosse virus [LACV] (genus Orthobunyavirus, family Peribunyaviridae) is a mosquito-borne virus, associated with clinical cases of pediatric encephalitis concentrated in the Midwest and Appalachian regions where it is also detected in mosquitoes during surveillance activities. Symptoms of La Crosse encephalitis include headache, fever, vomiting, and disorientation which may lead to seizure, coma, and death in severe cases [1]. Notable clusters of illness have occurred in Ohio, Wisconsin, Tennessee, and North Carolina with a substantial socioeconomic burden associated [1, 2, 3, 4]. In contrast, only one locally-acquired case of LACV has been reported in the northeastern USA during the last 20 years, occurring in upstate New York in 2010 and was non-neuroinvasive [5]. In addition, two human cases were reported in Rhode Island, during 2018 and 2019, but both were imported as these individuals had a travel history outside that state (Rhode Island DPH, personal comm.). LACV was rst detected in the New England region in a pool of mosquitoes collected in Connecticut in 2005 during statewide surveillance for arboviral pathogens [6]. Phylogenetic analysis indicated that the Connecticut isolate represented a genetically distinct lineage that diverged earliest from viruses circulating in other geographic regions of the USA [6, 7]. Despite the disease being associated within the Midwest and Appalachian regions of the USA, LACV may represent an unrecognized public health risk to residents of New England and New York. There is a need to understand the transmission cycles, prevalence, and strains of LACV in this region. Aedes triseriatus serves as the main vector of LACV which is broadly distributed throughout the eastern half of the USA [8]. Nevertheless, this species is likely under-sampled during statewide surveillance efforts because it does Page 2/16 not readily enter standard CDC light traps or gravid traps which are routinely used [3, 9]. Additional sampling and testing of Ae. triseriatus and other locally abundant mosquito species are needed to accurately estimate the entomological risk of LACV in enzootic sites. Here we report the results of mosquito monitoring activities detecting LACV in Connecticut. In 2018, we conducted two surveys that intensied the extent of mosquito sampling and testing in central and southwestern Connecticut and highlighted increased detection of LACV activity [10]. In addition, routine statewide surveillance in Connecticut, ongoing since 1997, detected four additional isolates of LACV since 2005. We present evidence for ongoing circulation of LACV in this region, and report on the entomological and viral phylogenetic data that supports local persistence of LACV in Connecticut. Methods Entomological data Mosquito Collections. Isolates of LACV analyzed in the present investigation were obtained from eld- collected mosquitoes that were procured from the following: a. Connecticut state has conducted annual monitoring of mosquitoes at 36 locations since 1997 and was increased to a total of 91 locations (8 counties) since 2001. Field surveillance was conducted using CDC light traps and gravid traps, and at some sites, BG Sentinel traps (Biogents, Germany), followed by mosquito identication and viral testing across the eight counties of Connecticut. Each site was sampled at least once every 10 days from June-October (Figure 1). We consider here, data from the 2005-2018 surveillance seasons. b. A trapping-lure comparison study (reported Eastwood et al, 2019) was conducted in Stamford, CT and Hamden, CT, capturing 33,649 and 14,085 individuals respectively using BG-Sentinel traps baited with C02 and different chemical lures. A total of 47,734 mosquitoes (32 species of 8 genera) were trapped and identied over 27 days of sampling in July and August 2018, and pooled for virus detection. c. Thirdly, focused sampling was performed during 2017 and 2018 at the location where LACV was rst detected in the region, (Faireld, CT; 41.19467, -73.32730), as well as two nearby sites - Easton, CT (41.28032, -73.30308) and Redding, CT; 41.30972, -73.32178). Here, CDC light traps, BG sentinel traps, MMX traps, and gravid traps, as well as GAT traps (Biogents) were deployed overnight at sites for two consecutive nights each week, August-October 2017 and May-October 2018 targeting adult mosquitoes. In addition, as a pilot study of vertical transmission at those sites, six oviposition cups (black plastic casino cups, lined with seed germination paper), were placed in the eld and checked weekly to collect container-breeding mosquito eggs. Mosquitoes captured within each study were sorted from other insect fauna, identied morphologically to species level using a key [11], with a cold-chain maintained throughout. Page 3/16 Virus detection Adult mosquitoes were identied and screened for arbovirus as follows. Pools of up to 50 mosquitoes (grouped by species, location, and capture date) are homogenized and inoculated on a Vero (African Green monkey kidney) cell line, for evidence of arboviral infection, as described Eastwood et al. 2019 [10]. Briey, mosquitoes were homogenized in a vial with 1 mL PBS-G (phosphate-buffered saline containing 0.5% gelatin, 30% rabbit serum, 1× antibiotic/antimycotic) and a copper BB pellet, using a mixer-mill set for 4 min 25 cycles/second. Samples were then centrifuged for 5 min at 7,000 rpm at 4°C. The supernatant (100 μl) was inoculated onto a conuent monolayer of Vero cells in 25-cm2 culture asks, allowed to absorb for 5 min on a plate rocker, then provided with 4 mL of minimum essential media supplemented with 10% fetal bovine serum, 1× antibiotic/antimycotic. Flasks were incubated at 37°C with 5% CO2 and examined daily for cytopathic effect (CPE) for up to 7 d. Infected cell cultures showing CPE were harvested and stored at −80°C. To identify viruses, RNA was extracted from isolates using a QIAampViral RNA mini kit (Qiagen, Germantown, MD), eluted in a nal volume of 70 μL. A reverse transcription polymerase chain reaction (RT-PCR) was performed in a 25 μL reaction using a Titan One- Tube RT-PCR system (Roche Diagnostics, Indianapolis, IN) with generic orthobunyavirus primers [10, 12]. Amplication products of the appropriate size, were puried as per Eastwood (2019), then commercially sequenced (Science Hill DNA Analysis Facility, Yale University, New Haven, CT). Mosquito eggs collected using the oviposition cups, as part of the third collection study, were reared to adults in the laboratory of the CAES at 25°C with 16/8 h light/dark photoperiod. This F1 generation was screened for virus as above, to test for evidence of LACV having been vertically transmitted. Minimum infection rates [MIR] of LACV were determined parsed by mosquito species/site/year using the CDC-provided Excel Add-in tool for calculating bias-corrected maximum likelihood estimate pooled infection rates [13] . Phylogenetic data Nucleotide sequencing and genetic analyses Viral RNA was isolated from virus cultures using the QIamp viral RNA Kit (Qiagen, Valencia, CA). RT-PCR was performed using the Titan One-Tube RT-PCR System (Roche Diagnostics, Indianapolis, IN) with primers targeting each of the three genomic segments of LACV. Primer pairs BUNS+new/BUNS-new and M14C/M4510r were used to amplify the entire S and M segments as previously described